The tumor suppressor gene PTEN is notable for two characteristics, first, being a direct inhibitor of the PI3-kinase (PI3K) growth stimulatory pathway via dephosphorylation of pathway products, phosphatidylinositol 3' phosphates. Second, in the majority of cancers including gliomas, PTEN appears to be preferentially altered in the high-grade form, suggesting its involvement in the malignant aspects of oncogenesis. A number of studies have shown that PTEN regulates the PI3K pathway, known to influence the growth, metabolism, migration and survival of cells. Our data suggest that while regulation of the PI3K pathway is essential for suppression of tumorigenicity, it is not sufficient, implying additional functions for PTEN. This is highlighted by two mutant forms of PTEN, Y315A and Y240A, which are tumorigenic when expressed in PTEN-deficient glioma cells, yet still able to mediate the activity of the PI3K pathway. Furthermore, we have recently made two highly interesting observations: 1) we have identified two proteins that specifically bind to a phosphorylated form of Y315, but not to the non-phosphorylated sequence; 2) subcellular localization of PTEN is cell cycle dependent. The major focus of this proposal is to define the additional signaling networks involving PTEN by characterizing their members, and to define the mechanisms that regulate PTEN's localization, activities and functions. Our data also suggest that PTEN activities are under strict regulation. We propose these activities are mediated, in part, by post-translational modifications (serine/threonine and tyrosine phosphorylations), associations with other proteins, and subcellular localization. The overall hypothesis of this study is that PTEN is a component of a multifunctional signaling complex whose function mediates biological and biochemical activities that are involved in the malignancy of tumor cells. The specific aims of this proposal are: 1) to delineate the molecular mechanisms involved in the regulation of PTEN's cell cycle-dependent subcellular localization. 2) to characterize the biological functions of PTEN in the nucleus and identify the downstream target genes specifically involved in nuclear PTEN-mediated growth suppression. 3) to identify and characterize proteins, which interact with PTEN to form a putative signaling complex, particularly proteins that interact with PTEN at phosphorylated tyrosine 315.